Recombinant eukaryotic viral vectors have become a preferred means of gene transfer for many researchers and clinicians. The human adenovirus is one of the most widely used recombinant viral vectors in current gene therapy protocols. As the use of adenoviral vectors becomes more prevalent, the need for systems that efficiently produce adenoviral vectors suitable for administration is increasingly important.
A concern associated with recombinant adenoviral vectors is uncontrolled propagation of the vector upon administration. To address this concern, replication-deficient adenoviral vectors, typically lacking the essential E1 region of the adenoviral genome, have been developed.
The production of replication-deficient adenoviral vectors is commonly accomplished by use of a complementing cell line, such as the 293 cell line developed by Graham et al. (J. Gen. Virol., 36, 59-72 (1977)), which provides in trans the gene functions lacking in the replication-deficient adenoviral vector. A problem associated with many complementing cell lines, including the 293 cell line, is the possibility of homologous recombination between the replication-deficient adenoviral genome and the regions of the adenoviral genome inserted into the complementation cell, resulting in production of replication-competent adenovirus (RCA). To reduce the frequency of RCA formation, several researchers have attempted to construct complementing cell lines comprising viral gene sequences that lack any homology to the adenoviral vector of interest (see, for example, International Patent Applications WO 94/28152 and WO 98/39411, and U.S. Pat. Nos. 5,994,128 and 6,033,908).
The construction of stable human cell lines that effectively and efficiently complement replication-deficient viral vectors can be difficult. For example, such cell lines often produce significant quantities of E1 and/or E4 gene products, resulting in undesired cytotoxic and/or cytostatic effects. High levels of E1A gene product expression, for example, induce apoptosis in host cells (Rao et al., PNAS, 89, 7742-7746 (1992)), while expression of E4 gene products induce p53-independent apoptosis in human cells (Marcellus et al., J. Virol.,72, 7144-53 (1998)). Thus, complementation cells, such as those known in the art, that constitutively express such factors may be associated with poor survival rates prior to and/or during adenoviral vector production.
Animal cells and other viruses encode gene products that are functionally homologous to adenoviral early region genes. Some of these gene products, when transiently expressed in human primary cells or non-human transformed cells, have been shown to complement for deficiencies in E1A gene functions. In particular, Tevethia et al., Virology, 161, 276-285 (1987), describes complementation in primary embryonic lung cells of an E1A-deleted adenoviral vector with plasmids encoding immediate early region genes of the human cytomegalovirus (CMV). In addition, the E7 protein of human papilloma virus 16 (HPV16) complements for deficiencies in the E1A region by immortalizing primary rat cells, as shown by co-infection experiments, while tamarin cells transformed by the Epstein-Barr virus (EBV) complement for deficiencies in the E1A and/or E2 regions (see, e.g., Kimura et al., Tumor Research, 32, 1-21 (1997), and Horvath et al., Virology, 184, 141-148 (1991)). Moreover, some cell lines, including the human hepatoblastoma line HepG2 and certain embryonic stem cell lines, encode factors that provide for the transcriptional transactivation function of the E1A region, as shown by activation of both E2A and/or E1B promoters in the absence of the E1A region (see, e.g., Spergel et al., Proc. Natl. Acad. Sci. USA, 88, 6472-6476 (1991), Spergel et al., J. Virol., 66, 1021-1030 (1992), Imperiale et al., Mol. Cell Biol., 4, 867-874 (1984), La Thangue and Rigby, Cell, 49, 507-513 (1987), and La Thangue et al., Nuc. Acids Res., 18, 2929-2938 (1990)).
Accordingly, there remains a need for alternative cells for propagating replication-deficient adenoviral vectors. The invention provides such cells. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.